Team:Dalton School NY/Notebook

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[[Image:Dalton_Fig8.jpg]]
[[Image:Dalton_Fig8.jpg]]
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Based on the data above, we concluded that the following clones had inserts of the correct size:
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'''Promoters'''
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MET15    yes: B
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GAL1    yes: A
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GAL1-L    yes: A
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GAL1-S    yes: B
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RAD52    yes: B
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RAD50    yes: A
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RAD54    yes: A, B
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RAD55    yes: A, B
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MRE11    yes: A, B
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XRS2    yes: A, B
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REV1    yes: B
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TDH3    yes: A, B
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LIF1    '''none'''
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MIG1    yes: B
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TUB1    yes: A
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CSE4    yes: B
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CLN2    yes: B
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CLB2    '''none'''
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ACT1    '''none'''
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ENO1    '''none'''
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ENO2    '''none'''
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TEF2    yes: B
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TEF1    '''none'''
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EAF6    '''none'''
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HSP12    yes: A, B
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HSP26    yes: A
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HSC82    '''none'''
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MSN2    yes: A
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MSN4    '''none'''
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HHO1    yes: A, D
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'''Protein-coding sequences'''
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BFP    maybe: A (the insert may be too large)
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GFP    '''none'''
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YFP    '''none'''
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mTangerine    '''none'''
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mCherry    yes: A (but bottom band seems possibly too intense)
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mGrape1    '''none'''
Return to our [https://2012hs.igem.org/Team:Dalton_School_NY main page].
Return to our [https://2012hs.igem.org/Team:Dalton_School_NY main page].

Revision as of 02:40, 1 June 2012

In progress...

Testing of DNA prepared by several methods

Yeast genomic DNA is commonly prepared using a phenol/chloroform based method. Because both phenol and chloroform are toxic chemicals, we tested several methods for preparing yeast genomic DNA that do not use these chemicals. We compared a method that uses LiOAc to disrupt the yeast cell wall (BioTechniques 50:325-328, 2011) to methods that use the enzyme zymolyase to disrupt the cell wall (a Promega kit or a protocol from Jim Haber’s lab, “JH zym”). The Haber lab protocol produced the greatest yield of genomic DNA, but DNA made using the Promega kit was used in subsequent PCRs because it seemed to produce the most consistent results. 1µl, 2µl, or 5µl of genomic DNA was used in each PCR reaction with LIF1 promoter primers. Following these initial tests, 1µl of yeast genomic DNA was used for subsequent PCRs. The LiOAc method was the shortest and least expensive and we may switch to this method when future DNA preps are needed.

Dalton Fig7.jpg

PCR of promoters and fluorescent protein coding sequences

Dalton Fig6.jpg

BsaI-digested Miniprep DNA

The samples on these gels contain BsaI-digested miniprep DNA prepared from individual colonies from the transformations above.

Dalton Fig8.jpg

Based on the data above, we concluded that the following clones had inserts of the correct size:

Promoters

MET15 yes: B

GAL1 yes: A

GAL1-L yes: A

GAL1-S yes: B

RAD52 yes: B

RAD50 yes: A

RAD54 yes: A, B

RAD55 yes: A, B

MRE11 yes: A, B

XRS2 yes: A, B

REV1 yes: B

TDH3 yes: A, B

LIF1 none

MIG1 yes: B

TUB1 yes: A

CSE4 yes: B

CLN2 yes: B

CLB2 none

ACT1 none

ENO1 none

ENO2 none

TEF2 yes: B

TEF1 none

EAF6 none

HSP12 yes: A, B

HSP26 yes: A

HSC82 none

MSN2 yes: A

MSN4 none

HHO1 yes: A, D


Protein-coding sequences

BFP maybe: A (the insert may be too large)

GFP none

YFP none

mTangerine none

mCherry yes: A (but bottom band seems possibly too intense)

mGrape1 none

Return to our main page.

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